Biochemical and Biological Properties of 4-(3-phenyl-[1,2,4] thiadiazol-5-yl)-piperazine-1-carboxylic acid phenylamide, a Mechanism-Based Inhibitor of Fatty Acid Amide Hydrolase

Abstract

Fatty acid amide hydrolase (FAAH) is an integral membrane enzyme within the amidase-signature family. It catalyzes the hydrolysis of several endogenous biologically active lipids, including anandamide (arachidonoyl ethanolamide), oleoyl ethanolamide, and palmitoyl ethanolamide. These endogenous FAAH substrates have been shown to be involved in a variety of physiological and pathological processes, including synaptic regulation, regulation of sleep and feeding, locomotor activity, pain and inflammation. Here we describe the biochemical and biological properties of a potent and selective FAAH inhibitor, 4-(3-phenyl-[1,2,4]thiadiazol-5-yl)-piperazine-1-carboxylic acid phenylamide (JNJ-1661010). The time-dependence of apparent IC(50) values at rat and human recombinant FAAH, dialysis and mass spectrometry data indicate that the acyl piperazinyl fragment of JNJ-1661010 forms a covalent bond with the enzyme. This bond is slowly hydrolyzed, with release of the piperazinyl fragment and recovery of enzyme activity. The lack of inhibition observed in a rat liver esterase assay suggests that JNJ-1661010 is not a general esterase inhibitor. JNJ-1661010 is >100-fold preferentially selective for FAAH-1 when compared to FAAH-2. JNJ-1661010 dose-dependently increases arachidonoyl ethanolamide, oleoyl ethanolamide, and palmitoyl ethanolamide in the rat brain. The compound attenuates tactile allodynia in the rat mild thermal injury model of acute tissue damage and in the rat spinal nerve ligation (Chung) model of neuropathic pain. JNJ-1661010 also diminishes thermal hyperalgesia in the inflammatory rat carrageenan paw model. These data suggest that FAAH inhibitors with modes of action similar to JNJ-1661010 may be useful clinically as broad-spectrum analgesics.

Computational insights into function and inhibition of fatty acid amide hydrolase

“…They showed that the decarbamoylation process of FAAH covalent adducts is favored for the piperazinylurea JNJ1661610 (10) [41], with respect to the Oaryl carbamate URB597 (6) [37]. This finding is consistent with the experimental observation of a slowly reversible FAAH inhibition for the piperazinylurea inhibitor and irreversible inhibition for URB597 (6) [33,122].…”

Chemical Probes of Endocannabinoid Metabolism

“…One dose of JNJ-1661010 (20 mg/kg i.p.) significantly blunted FAAH activity in the rat brain for up to 24 hours and caused a modest ;1.5-fold increase in brain anandamide levels (Keith et al, 2008;Karbarz et al, 2009). JNJ-1661010 administration elicited therapeutic analgesic effects in rat models of neuropathic, inflammatory, and acute thermal pain (Karbarz et al, 2009).…”

Enol Carbamates as Inhibitors of Fatty Acid Amide Hydrolase (FAAH) Endowed with High Selectivity for FAAH over the Other Targets of the Endocannabinoid System

“…At least 80 % of enzyme activity was recovered after dialysis of the enzyme-inhibitor complexes for 18 h (see Supporting Information figure S1). In contrast, dialysis of FAAH-URB597 complexes under similar conditions has shown that this agent is an irreversible inhibitor of the enzyme, [39] due to carbamoylation of Ser 241 in the active site. [40] Furthermore, nonlinear regression analysis of the Michaelis-Menten curves demonstrated that all of the selected compounds were noncompetitive inhibitors of FAAH with respect to the natural substrate AEA (see Supporting Information figure S2 A).…”

“…[39,40] The apparent K i values of compounds 1 b, 1 h, 1 i, 1 j towards FAAH are shown in Table 2. In a second set of experiments, we analyzed the interaction of compounds 1 b, 1 h, 1 i, 1 j with the other targets of the ECS, in order to calculate selectivity index values compared with FAAH (see Table 2).…”